KR20090114380A - Dna sequence encoding penicillin acylase, novel recombinant dna constructs and recombinant microorganisms carrying this sequence - Google Patents

Abstract

The invention consists in a nucleotide sequence having the size of (2646) bp, wherein the order of nucleotides is identical to the order of the nucleotide sequence encoding penicillin acylase from Achromobacter sp. CCM 4824 (formerly Comamonas testosteroni CCM 4824), eventually of the fragments of this sequence having the length of at least 150 nucleotides. The sequence can be used in the formation of a DNA construct, eventually the construct having at least one regulatory sequence regulating the expression of the gene and the production of a polypeptide with the penicillin acylase activity. The sequence can form part of a recombinant expression vector, which consists of the above-mentioned construct, promoter, translational start signal, translational and transcriptional stop signal. Further, the invention concerns a recombinant host cell, containing the nucleic acid construct carried by the vector or integrated into the cell chromosome, and the E. coli BL21 strain containing said sequence of the nucleotides encoding the penicillin acylase carried in the pKXIPl, the pKLP3 or the pKLP6 plasmid.

Description

DNA SEQUENCE ENCODING PENICILLIN ACYLASE, NOVEL RECOMBINANT DNA CONSTRUCTS AND RECOMBINANT MICROORGANISMS CARRYING THIS SEQUENCE}

The present invention relates to a DNA sequence encoding a polypeptide penicillin acylase, a novel recombinant DNA construct and a recombinant microorganism having the sequence.

Penicillin acylase (E.C. 3.5.1.11, penicillin amidohydrolase) is produced by bacteria, actinomycetes, fungi and yeast. These important industrial enzymes are divided into three groups based on their substrate specificities: penicillin G acylase (PGA), penicillin V acylase (PVA) and ester hydrolases of α-amino acids (AEH, conventionally). Named ampicillin acyla). Enzymes of the PGA family have a wide range of substrate specificities and catalyze the hydrolysis of the amide bonds of penicillin and cephalosporins. Of these, the following genera are among the bacterial production strains of PGA: Aeromonas, Achromobacter, Alcaligenes, Arthrobacter, and Bacillus ( Bacillus, Corynebacterium, Escherichia, Erwinia, Flavobacterium, Kluyvera, Micrococcus, Nocardia ), Proteus, Providencia, Pseudomonas, Sarrcina, Xanthomonas, Xylella (Process Biochem. 24: 146-154, 1989, Process Biochem) 27: 131-143, 1992, Biotechnol. Adv. 18: 289-301, 2000).

Apart from the production strain expressing PGA obtained by mutationsis, recombinant microorganisms containing a recombinant plasmid with a structural gene encoding PGA are Escherichia coli (CS 244 343 and CS 246 957). , Alcaligenes sp. Or fecalis (Current Microbiology 39: 2444-2448, 1999; EP 638649, Appl. Environ.Microbiol. 63: 3412-3418, 1997), Arthrobacter viscosus (Appl. Environ. Microbiol. 54: 2603-2607, 1988 a 55: 1351-1356, 1989; Gene 143: 79-83, 1994), Bacillus megaterium (J. Bacteriol. 93: 302-306, 1967, Appl Microbiol.Biotechnol. 25: 372-378, 1987; US 3 145 395; Process Biochemistry 29: 263-270, 1994), Kluyvera citrophila (Agric. Biol. Chem. 39: 1225-1232). , 1975; Gene 49: 69-80, Biotechnol. Letters 14: 285-290, 1992), Providencia rettgeri (Acta Biochim. Polonica 28: 275-284, 1981; J. Bacteriol. 168: 431-433, 1986; DNA sequences 3: 195-200, 1992).

Most prokaryotic strains of PGA are Gram-negative bacteria and the enzyme is located in the periplasma of the cell. In Bacillus megaterium culture, the enzyme is secreted from the cells into the medium.

The penicillin G acylase is used industrially for the hydrolysis of cephalosporins and phenylacetyl derivatives of penicillin G mainly for the preparation of intermediates 6-APA and 7-ADCA. Currently, these enzymes are also used in the synthesis reactions, in the acylation of the intermediates described above, leading to the production of semi-synthetic antibiotics (eg US 5 753 458 and US 5 801 011; WO 98/04732; WO 97/04086; Enzyme Microb.Technol. 25: 336-343, 1999; Synthesis of β-lactam antibiotics: Chemistry, Biocatalysis and Process Integration, Ed .: A. Bruggink, Kluwer Academic Publishers, Dordrecht / Boston / London, 2001 ).

For continuous and prolonged use of the PGA in catalyzing the enzyme reaction, the enzyme is stabilized by immobilization or encapsulation to form the enzyme catalyst. In this form, the enzyme exhibits higher pH stability, temperature stability and longer half-life under reaction conditions that catalyze the reaction process.

An object of the present invention is a nucleotide sequence of size 2646 bp, the nucleotide sequence having at least 95% identity with the nucleotide sequence shown in FIG. 5.

One aspect of the invention is also a fragment of the nucleotide sequence according to the invention, which has a length of at least 150 nucleotides, which encodes penicillin acylase.

Another aspect of the invention is a nucleic acid construct comprising a nucleotide sequence of the invention or a fragment of a nucleotide sequence of the invention, wherein at least one regulatory sequence modulates the expression of said gene and the production of a polypeptide having penicillin acylase activity. It is a nucleic acid construct having a (reguatory sequence).

Another aspect of the invention is a recombinant plasmid comprising a nucleotide sequence of the invention or a fragment of a nucleotide sequence of the invention.

Another aspect of the invention is a recombinant expression vector consisting of the nucleic acid construct, promoter, translation initiation signal, and translation and transcription stop signal of the invention.

Another aspect of the invention is a host cell comprising the nucleic acid construct of the invention. In the host cell, the nucleic acid construct may be maintained in the recombinant expression vector of the present invention or integrated into the cell genome.

Another aspect of the invention is the recombinant plasmids pKX1P1, pKLP3 and pKLP6, wherein the nucleotide sequences of the invention are inserted, isolated from strain Achromobacter sp., And characterized by restriction maps according to FIGS. 1 and 2.

Another aspect of the invention is strain Escherichia coli BL21 comprising the sequence of the invention carried by the plasmids pKX1P1, pKLP3 and pKLP6.

The basis of the present invention is a nucleotide sequence of size 2646 bp, the nucleotide sequence being the same nucleotide sequence as the nucleotide sequence shown in FIG. 5 and ultimately having a length of at least 150 bases that encodes penicillin acylase. Is a fragment of. The sequence may form part of a DNA construct, a recombinant plasmid and a vector. By means of a suitable vector, the sequence can be inserted into the genome of a bacterial or yeast host, which can then be used for the production of penicillin acylase.

One possible embodiment of the invention is the strain Escherichia coli BL21 (pKXlPl) (CCM 7394) comprising the recombinant plasmid pKX1P1, and the nucleotide sequence of the newly isolated structural gene having penicillin acylase activity Is made on the basis. The plasmid is characterized in that the DNA fragment having a size of 2646 bp (including the site having the SD sequence) is inserted into the plasmid vector pK19 (RD Pridmore, Gene 56: 309-312, 1987), and also shown in FIG. It features a restriction map. Preparation of the recombinant microorganism is strain Achromobacter ( Achromobacter ) sp. Isolation of chromosomal DNA from cells of CCM 4824 (originally Comamonas testosteroni CCM 4824; Plhackova et al., Appl. Microbiol. Biotechnol. 62: 507-516, 2003) and 5.1 kb fragments of the chromosomal DNA And recombinant microbial E. coli TOP10 (pKLP3). The nucleotide sequence of the structural gene pga was obtained by PCR technique (polymerase chain reaction) using the DNA of plasmid pKLP3. According to the determined NT sequence of the gene, DNA primers were designed (Table 1), which determined the entire nucleotide sequence of the gene, including the regulatory site, along with the SD sequence by the same PCR-sequencing technique. It was used to Based on the entire nucleotide sequence, primers have been devised that provide PCR-amplification of the total structural genes for penicillin acylase (PCR; W. Rychlik: Methods in Molecular Biology 15, 31-39, 1993). Strain Achromobacter sp. Chromosome DNA of CCM 4824 was used as template. The obtained PCR product was isolated and conjugated to a multicopy plasmid pK19 (RD Pridmore, Gene: 309-312,1987) to obtain a recombinant plasmid, followed by the host strain Escherichia coli TOPlO. (Invitrogen, USA) was used for transformation.

Preparation of competent cells and transformation of host strains with a number of recombinant plasmids are described in H. Hanahan, J. MoI. Biol. 166: 557-580, 1983. From grown colonies, recombinant plasmids were isolated by alkaline lysis method (HC. Birnboim a J. DoIy: Methods in Enzymology 100, 243-254, 1983), and the size and orientation of the inserted fragments ) Was determined.

In selected colonies of host TOP10, the activity of the enzyme penicillin acylase was tested by batch culture in LB medium. Recombinant plasmids isolated from strains with the highest overall activity of PGA were designated pKX1P1. From the plasmid construct, the pga gene was structurally expressed. The host strain BL21 was then transformed with the plasmid.

The resulting recombinant strain Escherichia coli with plasmid pKX1P1 and also producing penicillin acylase was incubated in a stirred bioreactor. The optimal procedure is to incubate the strain on mineral medium M9 supplemented with casein hydrolysate and glycerol as the carbon and energy source. Fed batch cultivation was performed at a temperature in the range of 20 to 30 ° C. while maintaining the pH in the range of 5.5 to 7.5 and maintaining the dissolved oxygen concentration in the medium at 10 to 40%.

1 shows a restriction map of recombinant plasmid pKX1P1.

2 shows the plasmids pKAGS au 401, pKLP3 and pKLP6.

FIG. 3 shows the transition of the optical density ((OD600)) and dry weight of cells (dry mass) of the culture during fed-batch culture of strain BL21 (pKXlPl) in a stirred bioreactor.

Figure 4 shows the trend of total activity (TA) and specific activity (SA) during fed-batch culture of strain BL21 (pKXlPl) in a stirred bioreactor.

5 shows the structural gene pga and the nucleotide sequence of the contiguous site.

Example 1

A. Culture of sp. and Isolation of Chromosome DNA

Achromobacter sp. Soil isolates of CCM 4824 (Skrob et al., Enzyme Microbiol. Technol. 32: 738-744, 2003; Plhackova et al., Appl. Microbiol. Biotechnol. 62: 507-516, 2003) were placed in 50 ml of LB medium. (g / g: tryptone 10, yeast extract 5, NaCl 5; pH 7.2-7.5) at 37 ° C. on a rotating shaker (200 rpm) for 12-16 hours. Biomass from 2 ml of culture was separated from the media by centrifugation, washed with saline solution and stored at -20 ° C.

Chromosomal DNA was isolated from de-frosted cells by the commercially available column GENOMIC-TIP 100 / G (Qiagen, Switzerland) and the appropriate buffer kit GENOMIC BUFFER SET (Qiagen).

Example 2

Recombinant DNA technology

DNA cleavage by restriction enzymes (Fermentas, Lithuania), analysis of DNA molecules on 1.0% agarose gel (USB, USA) in TBE buffer, ligation by T4 DNA ligase (Fermentas, Lithuania) It was performed according to standard protocol (J. Sambrook, 1989). For PCR reaction and DNA sequencing, primers (Table 1) were synthesized by MWG-Biotech AG (Germany) and Metabion (Germany). To perform the PCR reaction, Thermocycler PTC-200 (MJ-Research, Inc., USA) was used and all reactions were performed using Pwo SuperYield DNA polymerase in the presence of GC-RICH solution (ROCHE, Switzerland). . The size of the product of the PCR technique was determined on a 1% agarose gel in TBE buffer, where DNA of phage λ digested by restriction endonuclease Pst I was used as molecular weight standard. Certain products of PCR for further subcloning or sequencing were isolated from agarose gels using the QIAEX II Gel Extraction Kit (Qiagen, Germany), according to the manufacturer's instructions. The unique DNA fragments were purified with High Pure PCR Product Purification Kit (ROCHE, Switzerland), according to manufacturer's instructions. Preparation of competent cells and transformation of host strains are described in J. Hanahan, MoI. Biol. 166: 557-580, 1983, wherein soil culture medium Luria supplemented with antibiotic kanamycin (Km, 50 μg / ml) at 37 ° C. for 16 hours transformed competent cells with plasmids with the desired PCR fragments. 50 on Bertani (LB, g / 1: tryptone 10, yeast extract 5, NaCl 5, agar 15; pH 7.2-7.5) and finally supplemented with Km on an orbital shaker Gallenkamp (200 rpm) Cultured in ml of liquid LB medium. For subsequent analysis, recombinant plasmids were isolated using Qiagen Plasmid Midi Kit (Qiagen, Germany) and High Pure Plasmid Isolation Kit (ROCHE, Switzerland), according to the manufacturer's instructions. Determination of all NT DNA sequences was performed automatically at the Institute of Microbiology AS CR on a 3100 DNA Sequencer (Perkin-Elmer, USA). The obtained sequence was analyzed by software Lasergene (DNASTAR Inc., USA). Homology of NT sequences was confirmed by software BLAST (National Center for Biotechnology Information, USA; SF Altschul et al: Nucleic Acids Res. 25: 3389-3402, 1997).

Example 3

Nucleotide Sequencing and Expression of pga Gene

Strain Achromobacter sp. Chromosome DNA of CCM 4824 was partially cut with the enzyme Sau3 AI. The DNA- fragments of vector pK19 (RD Pridmore:. Gene 56 , 309-312, 1987) in the Bam HI- linear plasmid DNA (Bam HI-inearized plasmid DNA ) and was bonded (ligation). The resulting construct was then used for transformation of host strain E. coli TOPlO. Plasmid pKAG Sau 401 (FIG. 2) isolated from recombinant strain E. coli showing a penicillin acylase phenotype (PGA ⁺ ) was limited by Pst I. Subsequently, the largest P st I-fragment (approximately 5.1 kb) was subcloned into the P st I-linear vector pK19, resulting in two constructs, pKLP3 and pKLP6 with oppositely oriented P st I-insertions (FIG. 2). Formed. Recombinant strains E. coli TOPlO (pKLP3) and E. coli TOP10 (pKLP6) had the phenotype PGA ⁺ .

A separate plasmid and pKLP3 pKLP6, general-purpose (universal) M13 / pUC sequencing primers and subsequently derived from the pga] Using strictly specific primers (pga -strictly specific primers), used as the template for obtaining the complete nucleotide sequence of the pga gene (Primer list-see Table 1). The nucleotide sequence of the structural gene pga having 2592 nucleotides (including the stop triplet TAA) is, at the site defined by nucleotides 63 to 2592, the relevant microorganism Achromobacter xylosoxidans ssp. 92% identity to the pga gene of xylosoxidans (GenBank AF490005).

Based on the nucleotide sequence of the pga gene including the adjacent site obtained above, strain Achromobacter sp. Using the chromosomal DNA of CCM 4824 as a template, a primer was designed to enable PCR amplification of the structural gene pga . Inserted restriction sites Xba I, resp. From the site adjacent to the structural gene pga with Pst I, two designed primers UPACYL Xba 1 and REVACYL Pst 1 were used in a PCR reaction comprising the following steps: 1) 5 minutes at 94 ° C .; 2) 30 cycles of denaturation at 94 ° C. for 45 seconds, binding of primers at 60 ° C. for 45 seconds, and polymerization at 72 ° C. for 3 minutes; 3) Termination of polymerization at 72 ° C. for 10 minutes. Under these conditions, a specific PCR product of 2663 bp size was prepared having the entire structural gene pga comprising the preceding site of the regulatory site with the Shine-Dalgarno sequence. The pga -specific PCR product was cleaved with restriction endonuclease Xba I (target sequence in UPACYL Xba l primer), then conjugated to vector pK19 and cleaved with two polylinker enzymes Xba I and Sma I. . The resulting recombinant construct was referred to as the pKX1P1 plasmid (FIG. 1). DNA sequencing of the plasmid revealed that no insertion-deletion mutations occurred during the PCR-product insertion, but site mutation C → T was found at the 99th nucleotide of the structural gene pga compared to the originally assumed nucleotide sequence. It became. However, because the triplet does not change the amino acid form that it encodes, the mutation is silent. The prototrophic host strain E. coli BL21 (Invitrogen, USA) was transformed with the isolated recombinant plasmids pKLP6 and pKX1P1, to produce a prokaryotic expression system BL21 (pKXlPl) for PGA.

Example 4

Expression of pga in Escherichia coli

Preparation of Inoculum for Cultivation of Escherichia coli BL21 (pKX1P1) Strains

50 ml of LB medium supplemented with kanamycin (Km, 50 μg / ml) was inoculated from glycerol reserves stored at −70 ° C. (medium grown with glycerol according to J. Sambrook, 1989). The inoculum was incubated in an orbital incubator shaker Gallenkamp (200 rpm) at 28 ° C. for 16 hours.

Culture in Bioreactor

Strain BL21 (pKX1P1) was stirred in M9 medium (Table 2) supplemented with glycerol (5-25 g / 1) and casein hydrolyzate (5-25 g / 1) as carbon and energy sources under strictly limited conditions. Incubated in a bioreactor Biostat MD (B. Braun Biotech Intl., Melsungen, Germany): working volume 8.2 l, air flow rate 8-9 l air / min, initial agitation number 200-300 rpm, 20 At a temperature in the range from 28 [deg.] C. The pH of the medium was maintained in the range of 7.5 to 5.5, and the dissolved oxygen concentration (pO ₂ ) was automatically maintained in the range of 5 to 30% of the maximum oxygen saturation of the medium by adjusting the number of stirrings in the range of 200 to 840 rpm. . The incubation was carried out in fed batch culture for 20-25 hours (FIGS. 3 and 4). At the end of the culture, the following parameters were measured: cell concentration (cell dry weight, cdw), total activity (TA) and specific activity of penicillin acylase (SA).

The parameter was measured as follows:

Cell concentration: 28 g cdw / l in culture medium

Total activity: 18 000 U / l in culture medium

Specific activity: 670 U / g cdw

Enzyme Activity Measurement

For PGA enzyme activity determination, culture samples in volumes of 1-2 ml were taken from the production culture. The cells were separated by centrifugation, washed with 1-2 ml of distilled water, and further centrifuged and stored at -20 ° C. De-frosted cells were resuspended in 0.005 M phosphate buffer (pH 8.0). The activity of PGA was measured by titration using penicillin G as substrate at 37 ° C.

Components in Culture Medium M9 matter Chemical formula Concentration (g / L) Ammonium sulphate (NH ₄ ) ₂ SO ₄ 4.0 Potassium dihydrogen phosphate KH ₂ PO ₄ 13.6 Sodium chloride NaCl 10.0 Hydroxide hydroxide NaOH 3.0 Magnesium sulfate MgSO _{4 7} H ₂ O 2.0 Calcium chloride CaCl ₂ · 6H ₂ O 0.05 Ferrous sulphate FeSO ₄ 7H ₂ O 0.01

Recombinant strains of microorganisms comprising the nucleotide sequences of the present invention can be used in the production of penicillin acylase for various applications in the chemical and pharmaceutical industries.

<110> FERMENTA BIOTECH LIMITED          MIKROBIOLOGICKY USTAC AVCR v.v.i. <120> DNA SEQUENCE ENCODING PENICILLIN ACYLASE NOVEL RECOMBINANT DNA          CONSTRUCTS AND RECOMBINANT MICROORGANISMS CARRYING THIS SEQUENCE <130> PN0286 <150> PV 2007-82 <151> 2007-01-31 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 2646 <212> DNA <213> Artificial sequence <220> <221> misc_feature (222) (1) .. (2646) Achromobacter Species CCM 4824; Recombinant DNA <400> 1 cggagacaga ttcaatgaag cagcaatggt tgtcggccgc cctgttggcg gccagttcgt 60 gcctgcccgc gatggcggcg cagccggtgg cgccagccgc cggccagacg tccgaggcgg 120 ttgcggcacg gccccaaacc gccgatggca aggtcacgat ccggcgcgat gcctacggca 180 tgccgcatgt ctatgccgac acggtgtacg gcatcttcta cggctacggc tacgcggtgg 240 cgcaggaccg gctgttccag atggagatgg cgcggcgcag cacccagggc cgggtggccg 300 aggtgctggg cgcctcgatg gtgggcttcg acaagtcgat ccgcgccaat ttctcgcccg 360 agcgcatcca gcgccagttg gcggcgctgc cggccgccga ccgccaggtg ctggacggct 420 acgcggctgg catgaacgcc tggctggcgc gggtgcgggc ccagccgggc caactgatgc 480 ccaaggaatt caatgacctg ggtttcgcgc cggccgactg gaccgcctac gacgtggcga 540 tgatcttcgt cggcaccatg gccaaccgct tttcggacgc caacagcgag atcgacaacc 600 tggcgctgct gacggcgttg aaggaccggc atggcgccgc cgatgccatg cgcatcttca 660 accagttgcg ctggctgacc gacagccgcg cgccgaccac ggtgccggcc gaagcgggca 720 gctaccagcc gccggtgttc cagccggacg gcgcggaccc gctggcctac gcgctgccgc 780 gctacgacgg cacgccgccg atgctcgagc gggtggtgcg cgacccggcc acgcggggcg 840 tggtcgacgg cgcgccggcg acgctgcggg cgcaactggc cgcccaatac gcgcaatcgg 900 gccagcccgg catcgccggc tttccgacca ccagcaatat gtggatcgtg ggccgcgacc 960 acgccaagga cgcgcgctcg atcctgctga acggcccgca gttcggctgg tggaatccgg 1020 cctataccta cggcatcggc ttgcacggcg ccggcttcga cgtggtcggc aacacgccgt 1080 tcgcctatcc cagcattctg ttcggccaca atgcacacgt gacgtggggt tcgaccgcgg 1140 gcttcggcga tgacgtcgac atctttgccg aaaagctcga tcccgccgac cgcacgcgct 1200 atttccacga cggccaatgg aagacgctgg aaaagcgcac cgacctgatc ctggtgaagg 1260 acgcggcgcc agtgacgctg gacgtgtacc gcagcgtgca tggcctgatc gtcaagttcg 1320 acgacgcgca gcacgtggcc tacgccaagg cgcgcgcctg ggaaggctat gaactgcaat 1380 cgctgatggc ctggacccgc aagacgcaat cggccaactg ggaacagtgg aaggcgcagg 1440 cggcgcgcca tgcgctgacc atcaactggt actacgccga cgaccgcggc aacattggct 1500 acgcgcacac gggcttctat cccaggcgcc gtccgggcca cgatccgcgc ctgccggtgc 1560 ccggcaccgg cgagatggac tggctgggcc tgctgccgtt ctctaccaat ccgcaggtct 1620 acaacccgcg ccagggcttc atcgccaact ggaacaacca gccgatgcgc ggctacccgt 1680 ccaccgacct gttcgccatc gtctggggcc aggccgaccg ctacgccgag atcgagacgc 1740 gcctgaaggc catgaccgcg aacggaggca aggtcagcgc gcagcagatg tgggacctga 1800 tccgcaccac cagctacgcc gacgtcaacc gccgtcattt cctgccgttc ctgcaacgcg 1860 cggtgcaagg gctgccggcg gatgatccgc gcgtgcgcct ggtggccggc ctggcggcct 1920 gggacggcat gatgaccagc gagcgccaac cgggttactt cgacaacgcc ggcccggcgg 1980 tcatggacgc gtggctgcgc gccatgctgc ggcgcacgct ggccgacgag atgccggccg 2040 acttcttcaa gtggtacagc gccaccggct acccgacacc gcaggcgccg gccaccggtt 2100 cgctcaacct gaccaccggc gtcaaggtgc tgttcaacgc cctggccggg cccgaggctg 2160 gcgtgccgca gcgctatgac ttcttcaacg gcgcgcgcgc cgacgacgtc atcctcgcgg 2220 cgctggacga tgcgctggcg gcgctgcgcc aggcctatgg ccaggatccg gcggcatgga 2280 agatcccggc gccgccgatg gtgttcgcgc ccaagaactt cctgggcgtg ccgcaggccg 2340 acgccaaggc ggtgctgtgc tatcgggcca cgcagaaccg cggcaccgag aacaacatga 2400 cggtgttcga cggtaaatcg gtgcgcgcgg tggatgtggt ggcgccgggg cagagcggct 2460 tcgtcgcccc ggacggcacg ccgtcgccgc acacccgcga ccagttcgac ctgtacaaca 2520 ccttcggcag caaacgggtg tggttcacgg ccgatgaggt gcggcgcaac gctacgtcgg 2580 aagagacgtt gcgctacccg cggtaaggtc gcgcgcgccc tggtggctgg caagcctgca 2640 gtacac 2646  

Claims (10)

Nucleotide sequence of size 2646 bp, wherein the nucleotide sequence has at least 95% identity with the nucleotide sequence shown in FIG. A fragment of the nucleotide sequence of claim 1, having a length of at least 150 nucleotides, encoding penicillin acylase. A nucleic acid construct comprising a nucleotide sequence according to claim 1 or a fragment of a nucleotide sequence according to claim 2, wherein said nucleic acid construct has at least one regulatory sequence that regulates the expression of said gene and the production of a polypeptide having penicillin acylase activity. Nucleic Acid Constructs. A recombinant plasmid comprising a nucleotide sequence according to claim 1 or a fragment of a nucleotide sequence according to claim 2. A recombinant expression vector consisting of a nucleic acid construct according to claim 3, a promoter, a translation initiation signal, and a translational and transcriptional stop signal. A host cell comprising the nucleic acid construct of claim 3. 7. The host cell of claim 6, wherein said nucleic acid construct is maintained in a recombinant expression vector according to claim 5. 7. The host cell of claim 6, wherein said nucleic acid construct is integrated into said cell genome. Recombinant plasmids pKX1P1, pKLP3 and pKLP6, wherein the nucleotide sequences according to claim 1 are inserted, isolated from strain Achromobacter sp., And also characterized by restriction maps according to FIGS. 1 and 2. Strain Escherichia coli BL21 comprising the sequence according to claim 1 carried by the plasmids pKX1P1, pKLP3 and pKLP6 according to claim 9.

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